A number of polypeptides containing disulfide bonds are known to form trisulfide derivatives. Among these are superoxide dismutase, interleukin mutein and growth hormone (GH). In principle, however, other polypeptides containing disulfide bonds, e.g. insulin, interleukins and certain clotting factors (such as Factor VII) can potentially form trisulfide derivatives under specific conditions.
Among the most studied polypeptides known to form trisulfide derivatives are growth hormones (GH), in particular human growth hormone. The term “human growth hormone” (hGH; sometimes also referred to, inter alia, as “somatropin” or “somatotropin”) is generally understood to refer to the protein (polypeptide) hormone consisting of a single chain of 191 amino acid residues cross-linked by two disulfide bridges, the monomeric form thereof having a molecular weight of approx. 22000 (22 kDa). Growth hormone preparations isolated from human pituitary are not homogeneous. For example, a smaller (20 kDa) variant produced from the same gene is also known. The “basic hGH” variant (hGH-V) expressed by the placenta during pregnancy is another analogue/variant which is a product of a separate gene; like the 22 kDa hGH polypeptide it consists of 191 amino acid residues, but 13 amino acid residues at various positions in the sequence differ from those in 22 kDa hGH [see, e.g, Bewley et al., Adv. Enzymol. 42, 73-166 (1975), and Frankenne et al., J. Clin. Endocrin. and Metabol. 66, 1171-1180 (1988)].
In hGH (i.e. the 22 kDa polypeptide), four cysteine residues are present giving rise to the two disulfide bridges. The first disulfide bridge forms a major loop between Cys 53 and Cys 165, while the second one form a minor loop between Cys 182 and Cys 189. The minor loop is located at the molecule's surface, while the major loop is embedded within the hGH molecule. Trisulfide bridge formation can occur at both the minor and major loop.
Human growth hormone is essential, inter alia, for longitudinal bone growth and normal growth development during childhood. Numerous other effects or applications of hGH are known, including promotion of healing of bone fractures, breakdown of adipose tissue and minimization of post-operative fatigue syndrome.
Production of therapeutically useful polypeptides, e.g. hGH, on an industrial scale is currently usually accomplished by use of recombinant techniques. Some of the developed recombinant processes use bacterial hosts, such as different strains of Escherichia coli, while other processes use eukaryotic microorganisms, such as yeasts (e.g. Saccharomyces cerevisiae), as hosts.
After production (fermentation), the medium comprising the recombinant polypeptide is usually processed further. During such processing the polypeptide may be subjected to various extraction and purification processes, such as centrifugation, microfiltration and/or ultrafiltration.
One of the problems encountered using conventional recombinant manufacturing methods to produce polypeptides is the formation of polypeptide by-products. In the production of polypeptides containing disulfide bonds, the formation of derivatives, in particular trisulfide derivatives, can be problematical.
The trisulfide derivative in question is one wherein the peptide molecule contains an extra sulfur atom that forms a “trisulfide bridge” within the molecule. Such a trisulfide derivative has been isolated during production of growth hormone, where it constitutes an undesirable by-product.
Thus, WO 94/24157 discloses a method for detecting a hydrophobic derivative of hGH comprising an extra sulfur atom compared to native hGH. The extra sulfur atom of the hydrophobic derivative of hGH forms a “trisulfide bridge” yielding a hGH trisulfide derivative (TS-hGH). Further disclosed is a method for converting this hGH trisulfide derivative back to its native hGH form by treating the hGH trisulfide derivative with a mercapto compound, such as cysteine, glutathione, 2-mercaptoethanol or dithiothreitol.
In addition, the trisulfide derivative of hGH has been described by Andersson et al., “Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli,” Int. J. Peptide Protein Res. 47 (1996) pp. 311-321, and by A. Jesperson et al., “Characterisation of a trisulfide derivative of biosynthetic human growth hormone produced in Escherichia coli,” Eur. J. Biochem. 219 (1994) pp. 365-373 (1994), and trisulfide derivatives have also been reported for other peptides, viz. recombinant superoxide dismutase [Briggs et al., Biochem. Biophys. Acta, 537 (1978) pp. 100-109] and a mutein of interleukin [J. Breton et al., J. Chromatoqr. A 709 (1) (1995) pp. 135-46].
WO 96/02570 describes another method for converting the hGH trisulfide derivative back to the native form of hGH by treating the derivative with a sulfite compound, such as sodium sulfite, potassium sulfite or ammonium sulfite, or an alkaline-earth metal sulfite such as magnesium sulfite or calcium sulfite.
WO 00/02900 describes, inter alia, a method for the production of recombinant peptides with a low amount of trisulfides, characterized by the addition of a metal salt (e.g. potassium or sodium salt) during or after the fermentation step.
WO 04/31213 discloses, inter alia, a method for decreasing the amount of a trisulfide isoform impurity produced in recombinant production of a “growth hormone antagonist polypeptide” in genetically modified host cells, wherein the impurity is contacted with a “mercapto compound” (exemplified by sulfites, glutathione, β-mercapto-ethanol, dithiothreitol, cysteine and others). The application also discloses the use of chelating agents or metal salts to achieve a reduction in the amount of trisulfide formed.